This invention relates to plant protein purification. Specifically, the invention achieves the efficient separation of the functionally active plant glycoprotein, Miraculin, from the matrix of other constituents of vegetable matter in the berries of miracle fruits, using tandem hydrophobic interaction chromatography.
The taste-modifying properties of miracle fruits have been known to local people in West Africa for centuries, where the fruits have been used to sweeten sour foods and beverages such as koko and kenkey made from fermented maize and millet, and palm wine. The amazing properties of these fruits were first described in the scientific literature by F. W. Daniel (1) in 1852 who called it the `miraculous berry of West Africa`. The active ingredient in the berries which is responsible for this effect was isolated and identified as a glycoprotein and subsequently named Miraculin by Kurihara and Beidler(2). These researchers extracted the pulp of the berries in sodium carbonate buffer at high pH and subsequently purified the extract by ion-exchange chromatography using DEAE Sephadex A-25 and CM Sephadex C-25 columns. Almost simultaneously, Brouwer et al. (3) also reported an alternate method of isolation of the active ingredient based on extracting the pulp of the berries into solutions of highly basic compounds such as salmine and spermine, followed by ammonium sulfate fractionation and gel filtration chromatography.
These early investigators noted the peculiar difficulties in isolating Miraculin from the berries. According to them, the active ingredient was highly labile and subject to rapid degradation by proteolytic enzymes present in the berries. Secondly, like many plant proteinaceous materials, Miraculin is found together with other components in plant tissues with which it very closely associated particularly polyphenolic coloring agents such that it is extremely difficult to isolate the free glycoprotein. In order to solve the latter problem, Giroux and Henkin (4) proposed grinding the pulp of the berries with insoluble polyvinylpyrollidone (PVP) in order to adsorb the polyphenolic compounds before extraction into sodium carbonate buffer.
A more recent purification protocol for isolating Miraculin was developed by Theerasilp and Kurihara (5) based on extraction of the pulp with 0.5M sodium chloride solution, followed by ammonium sulfate fractionation. Further purification was accomplished by ion-exchange chromatography on CM-Sephadex column and affinity chromatography using concanavalin A-sepharose column. The initial extraction with sodium chloride at neutral pH in lieu of alkaline (high) pH extraction was intended to minimize loss of the active ingredient which is believed to be sensitive to high pH.
A further improvement to this method was proposed by Kamimura et al (6) to include the addition of various acidic buffer media to the extraction solution in order to protect and stabilize Miraculin during isolation. The recommended media include aqueous acidic buffers such as sodium acetate, sodium citrate, sodium phosphate, glycine-HCl, and sodium borate.
In summary, prior approaches suffer from the peculiar problems associated with isolation of Miraculin from the berries. For example, tannins and other polyphenolic compounds which are co-extracted with Miraculin interfere with its isolation. Miraculin is also subject to rapid degradation by proteolytic enzymes present in the berries. Prior methods require use of large quantities of ammonium sulfate for fractionation which has adverse environmental impact during waste disposal. Existing methods require cold room operations during the purification process, which increases the cost of process-scale operations.
The methods developed so far for purifying Miraculin have been limited to laboratory scale operations suitable for obtaining research quantities (milligrams to grams range) of the active ingredient. In order to produce commercial quantities of Miraculin, a robust process is required which can be readily scaled-up to industrial scale. Such a method should also incorporate modem cost-effective protein purification technologies which can be operated in tropical environments where the berries are cultivated and harvested. Advantageously such a method should also be applicable to purification of other plant proteinaceous material.